Reversed-phase electrochromatography, including reversed-phase ion exchange chromatography and reversed-phase electrophoresis, is a method of choice for the separation and detection of many types of solutes in multi-solute sample mixtures. The term "electrochromatography" is used herein to denote chromatography in which electroendosmotic flow is used to drive the mobile phase through a bed of separation media. This is distinct from chromatographic systems in which the mobile phase flow is driven by a pump or other pressure-applying device. Like any chromatography, however, peak resolution in reversed-phase electrochromatography is continually sought to be improved.
The present invention resides in two methods of improving peak resolution in reversed-phase electrochromatography. The first is by the use of a run buffer that contains a desorbing constituent whose concentration is raised after the sample has been loaded, and the second is by the use of a mobile phase containing an ionic or non-ionic surfactant at a concentration below its critical micelle concentration (CMC). The first method has a dual effect--it narrows the sample zone near the entry point of the sample into the separation medium, and it produces a gradient elution due to the gradual equilibration of the mobile phase to the higher concentration. Both effects result in a narrowing of the solute peaks as they emerge from the separation medium and/or are detected. The second method (the use of the surfactant) improves the partitioning of the solutes by increasing the ligand density and/or charge on the stationary phase.